Lipid compositions with high dha content

ABSTRACT

The invention provides lipid compositions comprising phospholipids having a high docosahexaenoic acid (DHA) content, which compositions are preferably extracted from natural sources. The lipid compositions are excellent sources of highly bioavailable DHA and they can be used in oral delivery vehicles, dietary supplements, functional foods, and the like.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Nos. 61/530,648 filed Sep. 2, 2011 and61/650,206 filed May 22, 2012, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Omega-3 fatty acids are often referred to as “essential” fatty acidsbecause they are needed for human health but are not sufficientlyproduced by the body alone. The two major health promoting omega-3polyunsaturated fatty acids are eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA). EPA and DHA are naturally found in certaincold-water fatty fish such as salmon, tuna, and mackerel. They can alsobe derived in the body from alpha-linolenic acid (ALA), which is anomega-3 fatty acid found in certain seeds, plant-based oils, andanimal-based oils. However, the body is very inefficient at convertingALA into EPA and DHA.

The modern diet is typically deficient in omega-3 essential fatty acidsand has become overloaded with pro-inflammatory omega-6 fatty acids,especially arachidonic acid. This heavy imbalance of omega-6 to omega-3fatty acids in the modern diet is thought to lead to an overallinflammatory state that contributes to several diseases. The increasedconsumption of vegetable oils and shortenings, beef, and dairy is one ofthe major reasons for the high amount of omega-6 fatty acids in the dietand the imbalance between omega-6 to omega-3 fatty acids. The NorthAmerican population, in particular, has among the lowest dietary intakeof omega-3 fatty acids in the world and the highest intake of thepro-inflammatory omega-6 fatty acids.

Adequate amounts of omega 3 fatty acids, including EPA and DHA, can beobtained in the diet from cold-water fatty fish such as salmon, tuna,and mackerel. However, larger fish species may contain high levels ofmercury, polychlorinated biphenyls (PCBs), dioxins or othercontaminants. Additionally, there is seasonal variability in the amountsof EPA and DHA found in these fish. Thus achieving an optimal andconsistent amount of omega-3 fatty acids through the intake of fishalone raises a number of safety concerns. Fatty acids supplements areavailable. However, conventional over-the-counter omega-3 fatty acidsupplements contain relatively impure material and are typically onlyabout 30% omega-3 fatty acids. Furthermore, omega-3 fatty acids insupplements are often provided as free fatty acids, ethyl esters, ortriglycerides, which may be inefficiently processed by the body. Thesenon-polar ingredients can induce burping and discomfort, and requirethat larger amounts be consumed to gain any beneficial effect because ofinefficiently processing.

Accordingly, there is a need for efficient processes for obtainingomega-3 phospholipids with high stability, good digestion properties, nosafety and environmental concerns, without “fishy odor” and which arehighly suitable for incorporation into dietary supplements, nutritionalsupplements and food products. There is also a need for new compositionsthat have a high ratio of omega-3 fatty acids compared to omega-6 fattyacids. There is further a need for compositions that include a highamount of DHA compared to EPA content.

SUMMARY

Docosahexaenoic acid (DHA) has important structural roles and alsounique anti-inflammatory roles in the body. Most sources of omega-3fatty acids are richer in EPA than DHA, therefore new sources of DHArich fatty acids are needed. The invention described herein providescompositions having high DHA content, methods of obtaining suchcompositions, and methods of using such compositions, for example, forthe treatment of various adverse conditions and diseases.

The invention thus relates to solid lipid compositions comprising a highcontent of EPA, n3-docosapentaenoic acid (n-3 DPA), and DHA. Thecompositions can be obtained from non-shellfish or non-crustaceansources. These lipid compositions are preferably extracted from naturalsources such as immature fish roe. The compositions are useful astherapeutic compositions, food additives, and the like. They can also becombined with carrier oils and/or other additives to provide furtheruseful products and therapeutic compositions.

Accordingly, the invention provides lipid compositions (e.g., a PL waxor PL oil) having high amounts of omega-3 phospholipids. A lipidcomposition can include about 50 wt. % to about 100 wt. % phospholipids.The omega-3 fatty acid content of the phospholipids can be at leastabout 25 wt. %, at least about 30 wt. %, at least about 40 wt. %, or atleast about 45 wt. %, of the total fatty acids of the composition. Forexample, the lipid composition can include about 25 wt. % to about 50wt. %, about 40 wt. % to about 50 wt. %, about 40 wt. % to about 55 wt.%, or about 35 wt. % to about 60 wt. %, of omega-3 fatty acids as apercentage of total fatty acids in the composition (e.g., about 27-32wt. % free fatty acid equivalents in the wax).

The fatty acids of the lipid composition can include high amounts of DHAand EPA. The lipid composition can include DHA in an amount of about20-40%, expressed as a percentage of total fatty acids in thecomposition and EPA in an amount of about 6-20%, expressed as apercentage of total fatty acids in the composition. In some embodiments,the lipid composition includes DHA in an amount of about 20-28%,expressed as a percentage of total fatty acids in the composition andEPA in amount of about 6-20%, expressed as a percentage of total fattyacids in the composition. In some embodiments, the DHA and EPA of thephospholipids can be present in a ratio of about 1.5:1 DHA:EPA to about3.5:1 DHA:EPA. In other embodiments, the DHA:EPA ratio can be about1.8:1 to about 2.5:1, or about 2.2:1 to about 2.8:1. The lipidcomposition can include less than about 1 wt. %, less than about 0.7 wt.%, or less than about 0.5 wt. %, of arachidonic acid.

The lipid composition can be a solid having a light brown or ambercolor. This solid phospholipid-rich composition can be referred to as aphospholipid wax, or PL wax. The lipid composition can also be asemisolid, or highly viscous oil (e.g., a PL oil), however the solidcompositions can be more stable due to lower oxygen penetration.

The lipid composition can be obtained from a fish composition, such asfish internal organs, fish portions after processing, or fish roe. Thefish roe can be mature fish roe or immature fish roe. The fish roe canbe, for example, immature herring roe, immature salmon roe, immaturemackerel roe, immature menhaden roe, or a combination thereof.

The lipid composition can be combined with other ingredients toformulate a dietary supplement, a nutritional supplement, a foodproduct, or an oral delivery vehicle, for example, for a supplement orpharmaceutical product.

The lipid composition can be combined with a carrier oil, for example,an oil from a source other than the source of the polar lipidcomposition. The different source can be vegetable oil, hill oil,microbial oil, fish oil, or a combination thereof. The combination ofthe lipid composition and carrier oil can be referred to as a MarineOmega-3 Phospholipid composition, or MOPL. The MOPL can be an oil thatis readily processed for a variety of uses.

The MOPL can include about 10 wt. % to about 90 wt. % of a PL wax or PLoil and about 10% to about 90% of a carrier oil. The carrier oil caninclude free fatty acids, mono-, di- and triglycerides, fatty acid ethylesters, or a combination thereof.

In some embodiments, the invention provides a composition that includesa first phospholipid composition extract from fish roe and a secondphospholipid composition extract from fish milt. A composition describedherein can also include a fish protein additive.

The invention thus provides a solid phospholipid-rich lipid compositioncomprising greater than about 60% phospholipids, said phospholipidshaving about 25-40% or about 35-50% w/w DHA and EPA attached thereto,said DHA and EPA present in a ratio of about 1.5:1 DHA:EPA to about3.0:1 DHA:EPA, wherein the lipid composition is a solid and issubstantially stable to oxidation.

The invention also provides an emulsion that includes a PL wax as or PLoil as described herein, and water, and optionally a carrier oil. Theemulsion can include, for example, about 0.1% to about 5% of the PL waxor MOPL.

In various embodiments, the invention provides a phospholipidcomposition (e.g., a PL wax or PL oil) as described above, wherein thephospholipids can comprise at least about 60 wt. %, at least about 65wt. %, at least about 70 wt. %, or at least about 75 wt. %, of thecomposition. The composition can be rich in phosphatidylcholine content.For example, phosphatidylcholine can be at least about 45 wt. %, atleast about 50 wt. %, at least about 54 wt. %, or about 55 wt. %, of thecomposition. Of the fatty acids in the composition, the fatty acids canhave a greater percentage of DHA than EPA. For example, the ratio of DHAto EPA can be at least 1.1:1, at least 1.2:1, at least 1.3:1, or atleast 2.5:1. Typically the ratio of DHA to EPA is at least about 1.5:1,up to about 3.5:1. The composition can also be low in arachidonic acid,for example, including less than about 10 mg/g, less than about 7 mg/g,less than about 5 mg/g, or less than about 4 mg/g. The composition caninclude about 10 ppm to about 1000 of Vitamin E, which can includevarious isomers of tocopherols and tocotrienols. The composition canalso be free of shellfish antigens.

The invention further provides a process for efficiently providing acomposition with high amounts of omega-3 phospholipids. The process caninclude contacting immature fish roe with a polar solvent; extracting alipid fraction from the immature fish roe, to provide a mainly polarlipid fraction comprising omega-3 phospholipids; and removing thesolvent from the lipid fraction comprising omega-3 phospholipids, toprovide a solid, semisolid or highly viscous, mainly polar lipidcomposition comprising omega-3 phospholipids, wherein at least about30%, at least about 40%, or at least about 50%, of the total fatty acidsare omega-3 fatty acids (with respect to the phospholipids of thecomposition, or with respect to the total composition, or both). Thesolid polar lipid composition can be a PL wax as described above. A PLwax or PL oil can be combined with a carrier oil to provide a MOPL.

The invention yet further provides a process of extracting aphospholipid-enriched lipid composition from a fish or fish by-product.The process can include mixing fish, fish roe, or a fish-by product witha carrier lipid either before or after contacting the fish, fish roe, orfish by-product with a polar solvent, to form a lipid carriercomposition; isolating a polar fraction from the carrier lipidcomposition; and removing solvent from the polar fraction to provide thephospholipid-enriched lipid composition.

The invention also provides a process of producing a solidphospholipid-rich lipid composition with a high content of DHA using themethods described herein, wherein the solid phospholipid lipidcomposition is substantially stable to oxidation. The invention alsoprovides a method of providing oxidative stability in an emulsioncomprising forming an emulsion with a PL wax or PL oil as describedherein. The emulsion can be formed with water and, for example, about0.1 wt. % to about 5 wt. % of the PL wax or PL oil.

The invention also provides methods of administering a polar lipidfraction or composition to a subject to provide a phospholipidcomposition to the subject, such as a phospholipid composition describedherein. A variety of diseases and conditions can be treated or inhibitedby administration of a phospholipid composition described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. An example of a phospholipid, according to one embodiment. Oneor the other fatty acid chain moiety and its associated carbonyl can beabsent to provide hydroxyl groups in various embodiments. The fatty acidchain moieties can be, for example, EPA, DHA, or a group recited inTable 2-5.

FIG. 2. An example of a process for obtaining a phospholipid compositionfrom a fish composition, according to one embodiment.

FIG. 3. An example of a specific process for obtaining a phospholipidcomposition from immature fish roe, according to one embodiment.

FIG. 4. An example of a process for preparing a phospholipid compositionby combining a phospholipid composition obtained from a fish compositionand a second lipid composition, according to one embodiment.

DEFINITIONS Definitions

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percents, proximate to the recited range that are equivalent interms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths, ortenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an effective amount can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a compound described herein, or anamount of a combination of compounds described herein, e.g., that iseffective to treat or prevent a disease or disorder, or to treat thesymptoms of the disease or disorder, in a host. Thus, an “effectiveamount” generally means an amount that provides the desired effect.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can include medical, therapeutic,and/or prophylactic administration, as appropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

A “fatty acid” refers to an alkanoic acid or an alkanoic acid moiety(i.e., the residue left after formal removal of the acid hydrogen),where the fatty acid includes at least about nine or ten carbon atoms.Non-limiting examples of fatty acids include decanoic acid (10:0),undecanoic acid (11:0), 10-undecanoic acid (11:1), lauric acid (12:0),cis-5-dodecanoic acid (12:1), tridecanoic acid (13:0), myristic acid(14:0), myristoleic acid (cis-9-tetradecenoic acid, 14:1), pentadecanoicacid (15:0), palmitic acid (16:0), palmitoleic acid (cis-9-hexadecenoicacid, 16:1), heptadecanoic acid (17:1), stearic acid (18:0), elaidicacid (trans-9-octadecenoic acid, 18:1), oleic acid (cis-9-octadecanoicacid, 18:1), nonadecanoic acid (19:0), eicosanoic acid (20:0),cis-11-eicosenoic acid (20:1), 11,14-eicosadienoic acid (20:2),heneicosanoic acid (21:0), docosanoic acid (22:0), erucic acid(cis-13-docosenoic acid, 22:1), tricosanoic acid (23:0), tetracosanoicacid (24:0), nervonic acid (24:1), pentacosanoic acid (25:0),hexacosanoic acid (26:0), heptacosanoic acid (27:0), octacosanoic acid(28:0), nonacosanoic acid (29:0), triacosanoic acid (30:0), transvaccenic acid (trans-1′-octadecenoic acid, 18:1), tariric acid(octadec-6-ynoic acid, 18:1), and ricinoleic acid(12-hydroxyoctadec-cis-9-enoic acid, 18:1) and ω3, ω6, and ω9 fatty acylresidues such as 9,12,15-octadecatrienoic acid (α-linolenic acid) [18:3,ω3]; 6,9,12,15-octadecatetraenoic acid (stearidonic acid) [18:4, ω3];11,14,17-eicosatrienoic acid (dihomo-.alpha.-linolenic acid) [20:3, ω3];8,11,14,17-eicosatetraenoic acid [20:4, ω3],5,8,11,14,17-eicosapentaenoic acid [20:5, ω3];7,10,13,16,19-docosapentaenoic acid [22:5, ω3];4,7,10,13,16,19-docosahexaenoic acid [22:6, ω3]; 9,12-octadecadienoicacid (linoleic acid) [18:2, ω6]; 6,9,12-octadecatrienoic acid(γ-linolenic acid) [18:3, ω6]; 8,11,14-eicosatrienoic acid(dihomo-γ-linolenic acid) [20:3 ω6]; 5,8,11,14-eicosatetraenoic acid(arachidonic acid) [20:4, ω6]; 7,10,13,16-docosatetraenoic acid [22:4,ω6]; 4,7,10,13,16-docosapentaenoic acid [22:5, ω6]; 6,9-octadecadienoicacid [18:2, ω9]; 8,11-eicosadienoic acid [20:2, ω9];5,8,11-eicosatrienoic acid (Mead acid) [20:3, ω9]; trans-10,cis-12octadecadienoic acid; cis-10,trans-12 octadecadienoic acid;cis-9,trans-11 octadecadienoic acid; and trans-9,cis-11 octadecadienoicacid. The acyl residues of a fatty acid moiety can also be conjugated,hydroxylated, epoxidized, and/or hydroxyepoxidized acyl residues.

An “omega-3 fatty acid” refers to a polyunsaturated fatty acid that hasthe final double bond in the hydrocarbon chain between the third andfourth carbon atoms from the methyl end of the molecule or moiety.Non-limiting examples of omega-3 fatty acids includeΔ-5,8,11,14,17-eicosapentaenoic acid (EPA),Δ-4,7,10,13,16,19-docosahexanoic acid (DHA) andΔ-7,10,13,16,19-docosapentanoic acid (n-3 DPA).

The term “phospholipid” as used herein refers to a glycerol phosphatewith an organic headgroup such as choline, serine, ethanolamine orinositol and either one or two fatty acids esterified to the glycerolbackbone. See FIG. 1. Phospholipids include, but are not limited to,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine andphosphatidylinositol as well as corresponding lysophospholipids. Forexample, a “phospholipid” can refer to an organic compound of Formula I:

wherein R¹ is a fatty acid residue or H, R² is a fatty acid residue orH, R³ is H or a nitrogen containing compound such as choline(HOCH₂CH₂N⁺(CH₃)₃OH⁻), ethanolamine (HOCH₂CH₂NH₂), inositol, or serine,and R⁴ is a negative charge, H, or a cation such as an alkali metalcation (for example, Li⁺, Na⁺, or K⁺). R¹ and R² are not simultaneouslyH. When R³ is H, the compound is a diacylglycerophosphate (also known asphosphatidic acid), while when R³ is a nitrogen-containing compound, thecompound is a phosphatide such as lecithin, cephalin, phosphatidylserine, or plasmalogen. The R1 site is referred to as position 1 of thephospholipid (per the stereospecific [sn] system of nomenclature), theR2 site is referred to as position 2 of the phospholipid (the sn2position), and the R3 site is referred to as position 3 of thephospholipid (the sn3 position). Phospholipids also include phosphatidicacid and/or lysophosphatidic acid. Sphingolipids containing a phosphorusgroup are grossly classified as phospholipids; they contain asphingosine base rather than a glycerol base.

The term “phospholipid wax” or “PL wax” refers to a mass ofphospholipids that is a solid at room temperature (˜23° C.). A PL wax asdescribed herein can have a melting point interval in the range of about28° C. to about 65° C. Some PL waxes can have a melting point intervalin the range of about 28° C. to about 38° C., about 28° C. to about 35°C., about 28° C. to about 34° C., about 35° C. to about 65° C., about40° C. to about 60° C., about 45° C. to about 55° C., or about 55° C. toabout 65° C. Other PL waxes can have a melting point interval in therange of about 50° C. to about 60° C., about 40° C. to about 50° C.,about 30° C. to about 40° C., about 30° C. to about 38° C., about 30° C.to about 35° C., or about 30° C. to about 33° C. The melting pointinterval can be an interval of about 2, about 3, about 4, about 5, about7, or about 10° C. within one of the recited ranges. The PL wax can bepliable and the solid can be dissolvable in oils such as vegetable orfish oils.

As used herein, a “PL oil” refers to a viscous oil derived from a PL waxwhere the PL wax is further processed or purified to provide the viscousoil, rich in phospholipids.

As used herein, the term “lipid composition” refers to a wax or oil thatcan be extracted from a fish composition, especially immature fish roe.The lipid composition typically contains about 60-70 wt. %, about 62-67wt. %, or about 63-66 wt. % of phospholipids, and certain neutral lipidsand other components, for example, as outlined in Table 2-2. As would bereadily recognized by one of skill in the art, the fatty acid profile ofthe phospholipids themselves will have a higher DHA and EPA content byweight than the overall lipid composition (e.g., a PL wax or PL oil)because of the presence of non-fatty acid components in suchcompositions.

The term “omega-3 phospholipid” as used herein refers to a tophospholipid molecule having an omega-3 fatty acid residue at the sn1position, the sn2 position, or both positions, of a phospholipidmolecule.

A composition having a “high amount” of omega-3 phospholipids refers toa composition where at least about 30 wt. % of the fatty acid groups onthe phospholipid are omega-3 fatty acid moieties. A high amount ofomega-3 phospholipid can also be a phospholipid composition where thefatty acid groups are the phospholipids are at least about 40 wt. % orat least about 50 wt. % omega-3 fatty acid groups, with respect to thetotal amount of fatty acids in the phospholipids of the composition.

The term “immature fish roe” as used herein refers to fish roe in whichthe eggs are primarily (e.g., greater than about 50%, greater than about60%, greater than about 80%, or greater than about 90%) meiotic prophasearrest prior to maturation caused by a hormonal signal. Immature herringroe refers to roe collected between late October and the end of January,for example, in Norwegian waters; whereas mature roe is collected inFebruary and March or later, for example, near the Norwegian coast. TheHjort maturity scale can be used to assess maturity of fish roe (see Hayet al., “Assessing and monitoring maturity and gonad development inPacific herring”; Can. Tech. Rep. of Fish and Aquat. Sci., Vol. 998;1981; Government of Canada, Fisheries and Oceans). In all teleosts,oocytes appear to undergo the same basic pattern of growth, regardlessof their reproductive strategy. The major developmental events occurringduring oocyte development can be broadly classified into six phases,according to the state of oocytes growth; they are: oogenesis, primaryoocyte growth, cortical alveolus stage, vitellogenesis, maturation andovulation. During the early stages of oocyte development, DNAreplication occurs (leptotene), homologous chromosomes pair (zygotene)and these pairs shorten and thicken (pachytene). The chromosomes thenunpair into lampbrush configurations (diplotene), just before the oocyteenters a long period of cytoplasmic growth. The cytoplasmic growth ofthe oocyte is characterized by an enormous accumulation of yolk reserves(vitellogenesis). Meiosis resumes via a hormonal signal, and this leadsto oocytes maturation. During this period, the nucleus, arrested inmeiotic prophase, breaks down and the chromosomes enter first meioticmetaphase. The oocyte is then released from the ovary into the bodycavity and it becomes an egg ready for fertilization. Generally,immature roe is roe prior to ovulation or swelling.

The terms “subject” and “patient” refer to any animal, particularly amammal such as a dog, a cat, a bird, livestock, or a human.

The term “physiologically acceptable carrier” refers to any carrier orexcipient commonly used with pharmaceuticals, and especially for oraldelivery. Such carriers or excipients include, but are not limited to,oils, starch, sucrose and lactose as well as excipients for otherdelivery methods such as topical and parenteral delivery.

The term “oral delivery vehicle” refers to any means of delivering adesired composition orally, including, but not limited to, capsules,pills, tablets and syrups.

The term “emulsion” refers to a mixture of two or more liquids that arenormally immiscible (non-mixable or unblendable, i.e., they do not forma solution). Emulsions are part of a more general class of two-phasesystems of matter called colloids. The term emulsion is used when boththe dispersed and the continuous phase are liquids, such as water and anoil, for example, a combination of a PL wax and a fish oil or other highDHA oil. In an emulsion, one liquid (the dispersed phase) is dispersedin the other (the continuous phase), to provide an oil-in-water (o/w)emulsion or a water-in-oil (w/o) emulsion. The phospholipid compositionsdescribed herein are typically provided as o/w emulsions. However, whenusing very high amounts of the phospholipid composition relative towater, a w/o emulsion can be prepared.

The term “food product” refers to any food or feed suitable forconsumption by humans, non-ruminant animals, or ruminant animals. The“food product” may be a prepared and packaged food (e.g., mayonnaise,salad dressing, bread, or cheese food) or an animal feed (e.g., extrudedand pelleted animal feed or coarse mixed feed). “Prepared food product”means any pre-packaged food approved for human consumption.

The term “foodstuff” refers to any substance fit for human or animalconsumption. Examples include solid food, liquid food, combinationsthereof, and beverages.

The term “dietary supplement” refers to a small amount of a compound forsupplementation of a human or animal diet packaged in single or multipledoes units. Dietary supplements do not generally provide significantamounts of calories but may contain other micronutrients (e.g., vitaminsor minerals).

The term “nutritional supplement” refers to a composition comprising a“dietary supplement” in combination with a source of calories. In someembodiments, nutritional supplements are meal replacements orsupplements (e.g., nutrient or energy bars or nutrient beverages orconcentrates).

The term “functional food” refers to a food product to which abiologically active supplement, such as a phospholipid compositionsdescribed herein, has been added. A functional food is a food that hasdietary or nutritional components at a higher amount of % RDA thannaturally occurring food. A functional food can be aphospholipid-fortified medical food or conventional food.

The condition “dermatitis” refers to an inflammation of the skin such asa rash. Various forms of dermatitis may have common symptoms such as anallergic reaction to specific allergens. The term can include eczema,also referred to as dermatitis eczema and eczematous dermatitis. Aneczema diagnosis can imply atopic dermatitis, common in children andteenagers. Dermatitis can refer to an acute condition whereas eczemarefers to a chronic condition.

The condition “psoriasis” refers to an erythemo-scaly dermatitis ofchronic evolution. Psoriasis is often characterized by thickened patchesof reddish skin covered by slivery-white scales. The condition affectsan estimated 2 percent of the population. With approximately 60,000 newcases diagnosed each year, it is one of the most prevalent skindiseases. It commonly appears as painful itching, cracked, and/orbleeding skin, which can affect over 10 percent of the body surface. Itcommonly manifests itself on the elbows, on the ulnar edge of theforearms, on knees, lower back, scalp, and nails. Severe psoriasis isknown as erythrodermic psoriasis, psoriatic arthritis or pustularpsoriasis. About 1.5 percent people develop psoriasis during childhood,before age 10, and 35 percent before age 20. This abnormality of theepidermis renewal is thus a serious affection, representing an importantPublic Health concern.

DETAILED DESCRIPTION

Omega-3 phospholipids are an important alternative to free fatty acid,methyl- and ethyl ester and triglyceride forms of omega-3 because theyprovide essential fatty acids in a more bioavailable form and they areprocessed by the body in a more efficient manner. However, currentmethods of obtaining phospholipids do not provide the high andconsistent purity and high docosahexaenoic acid (DHA) content that isobtainable by the methods described herein. The invention thus providesefficient processes for obtaining omega-3 phospholipids with highstability, good digestion properties, no safety and environmentalconcerns, without “fishy odor” and which are highly suitable forincorporation into dietary supplements, nutritional supplements and foodproducts.

It is well established in the art that the omega-6 fatty acid,arachidonic acid, competes for incorporation with omega-3 fatty acidsfor incorporation into phospholipid and other pools of lipids,diminishing the effectiveness of omega-3 fatty acids. For an omega-3dietary preparation to be effective, the preparation must thereforecontain very low levels of arachidonic acid, such as less than 1 wt. %of the total fatty acids in the product. Chicken egg-derivedphospholipids and chicken egg-derived phosphatidylcholine providephospholipids and the valuable nutrient choline, but do not provideappreciable levels of DHA and EPA, and unfortunately contain about 3 wt.% of arachidonic acid.

Another key advantage of phospholipid forms of omega-3 fatty acids overtriglyceride forms of omega-3 fatty acids is that the former providesthe key nutrient choline, which is lacking in many diets and contributesto proper liver function and reduction in homocysteine, acholesterol-raising molecule. The importance of dietary choline isrecognized by a claim permitted by the European Food Safety Authority(EFSA). Choline may be obtained in the diet, or converted fromphosphatidylethanolamine in predominately liver cells via the enzymephosphatidylethanolamine N-methyltransferase (PEMT). Bothphosphatidylcholine and phosphatidylethanolamine, in specific ratios,are necessary to prevent fatty liver (steatosis or hepatosteatosis).

With increases in obesity, diabetes, and metabolic syndrome, steatosisis reaching epidemic proportions, and there are not effective drugtreatments at this time. Steatosis can impair liver function, andprogress to a further inflammatory condition of the liver known asnon-alcoholic steatohepatitis (NASH). NASH in turn progresses tofibrosis then cirrhosis. Liver-related death is in fact a leading causeof mortality. Adequate supplies of dietary phosphatidylcholine andphosphatidylethanolamine can be of benefit in preventing and treatingsteatosis and the down stream sequelae described above.

Post-menopausal women and individuals with specific PEMT polymorphismsare unable to synthesize phosphatidylcholine adequately fromphosphatidylethanolamine, and tend to have lower levels of hepaticphosphatidylcholine enriched with DHA. Thus, in these susceptiblepopulations, it is particularly important that they receive adequatephosphatidylcholine enriched with DHA as present in wax PL and MOPL. Asdescribed above, both phosphatidylcholine and phosphatidylethanolamineare needed to prevent fatty liver. Moreover, phosphatidylethanolamineenriched with DHA is known to be a predominant source of hepaticphosphatidylcholine enriched with DHA, after PEMT conversion. Thus, itis desirable for the general population to receive DHA-enriched in bothphosphatidylcholine and phosphatidylethanolamine, such as can beprovided by the PL wax and MOPL.

Food allergies are a growing public health concern, with an estimated 9million, or 4%, of adults in the United States having food allergies.The prevalence of food allergies and associated anaphylaxis appears tobe on the rise. According to a study released in 2008 by the Centers forDisease Control and Prevention, an increase in food allergy of about 18%was seen in the United States between 1997 and 2007. Eight foods accountfor 90% of all food-allergic reactions. These include milk, eggs,peanuts, tree nuts, wheat, soy, fish, and shellfish. Allergic reactionsto shellfish are generally a lifelong allergy. The estimated prevalence,some based on self-reporting, among the U.S. population is 0.4% for fishand 1.2% for crustacean shellfish. Shellfish allergies are likelyunderestimated because those sensitive to crustaceans may also besensitive to mollusks and/or /bivalves. In view of these data, the idealphospholipid source of omega-3 fatty acids should be fish (roe, milt,body)-derived, rather than shellfish/crustacean/krill derived.

The invention thus relates to lipid compositions comprising a highcontent of DHA, which is preferably extracted from a natural source.Surprisingly, the inventors have found that stable, solid,phospholipid-rich lipid compositions with both a high phospholipidcontent and a high omega-3 fatty acid content, particularly enriched inDHA, can be efficiently prepared from fish and fish by-products,including immature fish roe, mature fish roe, milt and internal organsof fish. See for example, FIG. 2. The high omega-3 fatty acid content ofthe extracted phospholipids can include particularly high levels of EPA(eicosapentaenoic acid (20:5 (n-3))) and DHA (docosahexaenoic acid (22:6(n-3))).

As discussed above, a PL wax can be extracted from a fish or fishby-product. In some embodiments, the fish is a cold water pelagic fish.In some embodiments, the fish by-product comprises fish roe. In someembodiments, the fish roe is immature fish roe. In some embodiments, thefish roe is mature fish roe. In some embodiments, the fish by-product ismilt. In some embodiments, the fish by-product comprises fish internalorgans. In some embodiments, the product is a mixture of roes, milts,and internal organs. The lipid composition can be substantially stableto oxidation. The stability of a composition to oxidation can bemeasured by accelerated shelf life tests, with oxidation determinedusing standard indices such as the amount of non-oxidized fatty acidsremaining, and the presence of thiobarbituric acid reactive substances(TBARS), and peroxide value (PV) of the composition.

The solid polar lipid extracted from fish roe is known as “wax” or a “PLwax”. Wax from mature and immature herring roe can contain phospholipidsat about 60-70% by weight, with almost no variation related to maturityin the phospholipid content and the omega-3 fatty acid content. The PLwax can contain about 30-65% by weight of the long chain omega-3 fattyacids EPA, DHA, and n3 DPA, typically esterified to phospholipids,predominately phosphatidylcholine, with a typical ratio of DHA:EPA of2-3:1. In various embodiments, the DHA and EPA can be present in a ratioof about 1.5:1 DHA:EPA to about 3:1 DHA:EPA, for example, about 1.7:1DHA:EPA, about 1.8:1 DHA:EPA, or 2.5:1 DHA:EPA.

To produce a liquid oil product, the PL wax may be blended with acarrier oil. The carrier oil can be a vegetable oil, a fish oil, a krilloil, a microbial oil, or a combination thereof. The microbial oil can bean algal oil, a fungal oil, or the like, or a combination thereof. Thecarrier oil can also be a natural oil, or a chemically- orgenetically-modified oil. The carrier oil can have a DHA:EPA ratiosimilar to the PL wax (i.e., +/−10% or 20%). In some embodiments, theprocess further comprises formulating a PL wax or a PL oil in an oraldelivery vehicle, a topical delivery vehicle, a sub-lingual deliveryvehicle, or a parenteral delivery vehicle.

In some embodiments, the oral delivery vehicle is a gel capsule. Theoral delivery vehicle can be, for example, a chewable gel. In otherembodiments, the invention provides a functional food product comprisinga lipid composition described herein.

By blending the PL wax or PL oil with a carrier oil with a high DHAcontent, the ratio of DHA:EPA of 2-3:1 can be maintained, whileproviding a final product that is less viscous. Typical blends of a PLwax and fish oil (e.g., a MOPL) include 0.74-9 parts PL wax: 1 part fishoil. In one embodiment, the MOPL or “designer oil” can be prepared bycombining about 10% to about 90% w/w of a PL wax and about 10% to about90% of a second lipid fraction or “carrier oil”. The carrier oil caninclude triglycerides, fatty acid ethyl esters, free fatty acids, or anycombination thereof. Proper selection of the type, viscosity, andDHA:EPA content of the carrier oil enables one of skill in the art toprovide a final product tailored to have certain physical andbiochemical properties for specific purposes; other additives can beincluded in the designer oil such as conjugated linoleic acid,carnitine, and the like. For example, the carrier oil can be selectedfor certain desired properties such as fluidity, viscosity and overallfatty acid content (thereby forming a “designer oil” or “tailored oil”).Thus, the carrier oil can contain a high content of EPA or other desiredfatty acid so that the level of EPA or other desired fatty acid in thecomposition can be increased as compared to DHA, or vice versa.

In some embodiments, a polar lipid fraction is extracted from a fishcomposition, such as immature fish roe, using a polar solvent such asethanol. A carrier liquid can then be added to the polar lipid fractionand polar solvent to provide a carrier-enriched composition, forexample, when the carrier is a fish oil or vegetable oil. Alternatively,water can be added to the polar lipid fraction and polar solvent tocreate an emulsion. Various amounts of ethanol may need to be removedbefore an effective emulsion can be prepared. The emulsion can include,for example, about 0.1% to about 5% of the PL wax, PL oil, or MOPL. Inother embodiments, the emulsion includes about 0.2% to about 2.0% of thePL wax or PL oil. In yet other embodiments, an emulsion can be preparedwith any amounts of PL wax and water, for example, varying from 1 partPL wax and 19 parts water, to 19 parts PL wax and 1 part water. Suchemulsions thus provide a range of qualities and properties, and w/oemulsions as well as o/w emulsions. The PL wax can increase theoxidative stability of the emulsion, and in particular the oxidativestability of other lipids in the emulsion. Accordingly, in someembodiments, the invention provides methods for improving oxidativestability in an emulsion by forming an emulsion with a PL wax or PL oilas described herein.

The inventors have discovered that omega-3 phospholipids extracted fromimmature fish roe, a previously underutilized natural resource that ispart of an existing fishery, have a desirable content of omega-3 fattyacids as described above, as well as having desirable physicalproperties that facilitate use for oral delivery and in nutritionalsupplements, dietary supplements, and food products. The omega-3phospholipids can be, for example, extracted from immature fish roe,mature fish roe, milt, and internal organs. The invention is not limitedto extraction of omega-3 phospholipids from any particular immature fishroe. However, in some embodiments, the immature fish roe is fromherring, mackerel, menhaden, or salmon.

Extraction of omega-3 phospholipids may be accomplished by a variety ofmethods. For example, a polar solvent can be used for the extraction. Insome embodiments, the polar solvent is ethanol. In other embodiments,the omega-3 phospholipids are extracted by super critical fluidextraction, preferably with a polar entrainer such as ethanol.Extraction with a polar solvent yields a polar lipid fraction that isenriched for omega-3 phospholipids. The solvent can be removed from thepolar lipid fraction to provide a solid polar lipid composition that isenriched for omega-3 phospholipids. In various embodiments, extractionis by supercritical fluid extraction (SFE) using CO₂ as a solvent andethanol as a polar entrainer. Some useful techniques for SFE aredescribed by U.S. Patent Publication No. 2011/0160161 (Sampalis et al.).

The lipid compositions described herein are excellent source of DHA andEPA. The solid phospholipid-rich lipid compositions can also be pliable,and have a light brown or amber color. The solid phospholipid-rich lipidcompositions are stable for extended periods of time, and for periods oftime longer than corresponding fatty acids without phospholipidmoieties, due to the antioxidant properties of the phospholipidmoieties. The phospholipid compositions are highly suitable forformulation or incorporation into oral delivery vehicles, dietarysupplements, nutritional supplements and food products. Accordingly theinvention also provides processes for incorporating or formulating thelipid compositions described herein into desired products, such as thosedescribed below.

The invention also provides a PL wax, PL oil, or MOPL composition foradministration to a subject, for example, to treat a condition ordisease described herein. The polar lipid composition can also be usedto improve the cognitive functioning of a subject. In other embodiments,the polar lipid fraction or composition is administered to a subject totreat or inhibit high blood triglycerides, high cholesterol,inflammation, hypertension, metabolic syndrome, obesity, otherconditions recited herein, or a combination thereof.

Phospholipid Compositions

The invention provides various phospholipid compositions that haveadvantageous amounts and ratios of fatty acid esters. A phospholipidcomposition such as MOPL can include a combination of a triglyceride(e.g., a fish oil) and an extracted polar lipid rich in phospholipids(e.g., a PL wax). The combination can be present, for example, in aratio ranging from about 1:10 to about 10:1. The phospholipids can havea structure such as Formula I:

wherein R¹ is H or a fatty acid moiety, R² is H or a fatty acid moiety,each R³ is independently H, or a choline, an ethanolamine, an inositol,a glycerol (as in phosphatidylglycerol or diphosphatidic acid), or aserine moiety, and R⁴ is a negative charge, H, or a cation such as analkali metal cation (for example, Li⁺, Na⁺, or K⁺). In some embodiments,the phospholipid can have DHA and/or EPA moieties as at least 1% of theR¹, R², and R³ groups. The phospholipids can have an overallconcentration of OH groups on structures of Formula I in the range ofabout 2.5% to about 80%, about 5% to about 70%, about 10% to about 60%,or about 20% to about 50%.

In some embodiments, the omega-3 fatty acids moieties include EPA, DHA,n-3 DPA, and/or α-linolenic acid (ALA). In various embodiments, thecomposition is substantially free of organic solvents and volatileorganic compounds such as short chain fatty acids, short chain aldehydesand short chain ketones (e.g., where short chain refers to C₁-C₆ alkyl).

In some embodiments, the phospholipid has at least 5%, or at least 10%,by weight, of a combination of EPA and DHA moieties esterified to theglycerol backbone. In various embodiments, the phospholipid has at least20% of a combination of EPA and DHA moieties esterified to the glycerolbackbone. The phospholipid can also have at least 30% of a combinationof EPA and DHA moieties esterified to the glycerol backbone. In yetother embodiments, the phospholipid contains about 5%, about 10%, about20%, about 30%, about 40%, or about 50% EPA/DHA moieties attached toposition 1 and/or position 2 to the glycerol backbone. The remainder ofthe groups can be, for example, other fatty acids described herein orhydroxyl groups. In some embodiments, the phospholipid has a ratio ofDHA:EPA ranging from about 1:1 to about 4:1. In other embodiments, thecomposition has a ratio of DHA:EPA ranging from about 2:1 to about 4:1.In other embodiments, fractionation and purification techniques can beused to produce a composition with a ratio of DHA:EPA of 10:1 orgreater.

In some embodiments, glycerol oxygen atoms that are not phosphorylatedin the composition can be about 30% to about 99% acylated, or about 40%to about 96% acylated. In other embodiments, the composition is acylatedin a range from 50% to 78%. In yet other embodiments, the phospholipidsare acylated to a degree of about 50% to about 90%, about 55% to about85%, about 60% to about 80%, about 40% to about 80%, or about 55% toabout 75%.

In some embodiments, the phospholipid extracted from fish roe can be asolid, such as a PL wax. In other embodiments, the phospholipidextracted from fish roe can be an oil, such as when the extractionprocess is modified to obtain only certain compounds or groups ofcompounds, for example, a PL oil.

The composition can be a marine lipid composition formulated into ananimal feed, a food product, a food supplement, a drug delivery system,or a drug.

The composition that include the phospholipid can further include alipid carrier. The lipid carrier can be present, for example, in a ratioof from 1:10 to 10:1 to PL wax or PL oil. In some embodiments, the lipidcarrier and the PL wax or PL oil can be present in a ratio of about 5:1to 1:5. In various embodiments, the composition includes about 20% toabout 90% of the wax and about 10% to about 80% of the lipid carrier.

The various embodiments are not limited to any particular lipid carrier.In some embodiments, the lipid carrier is a triglyceride, a diglyceride,a monoglyceride, an ethyl ester, and a methyl ester, or a combinationthereof.

In some embodiments, the invention provides methods of preparing abioavailable omega-3 fatty acid composition. The methods can include a)providing a phospholipid composition that includes omega-3 fatty acidresidues and optionally a triglyceride composition that includes omega-3fatty acid residues; and b) combining the phospholipid composition andthe triglyceride composition to form a bioavailable omega-3 fatty acidcomposition. The bioavailable phospholipid composition can be one of thecompositions described above. In some embodiments, the methods furthercomprise encapsulating the bioavailable omega-3 fatty acid composition.In various embodiments, the bioavailable omega-3 fatty acid compositionprovides increased bioavailability compared to purified triglycerides orphospholipids comprising omega-3 fatty acid residues. The methods canfurther include packaging the bioavailable omega-3 fatty acidcomposition for use in compositions such as functional foods. Themethods can also include assaying the bioavailable omega-3 fatty acidcomposition for bioavailability. In some embodiments, the methodsfurther include administering the bioavailable omega-3 fatty acidcomposition to a patient. The invention also provides a food product,animal feed, food supplement or pharmaceutical composition made at leastpartially by one or more of the foregoing process.

In some embodiments, the composition can include a useful nutritionalamount of cholesterol. Some compositions, such as a MOPL 50 product, caninclude about 3-10 g, about 4-8 g, about 4-6 g, about 6-8 g, about 5 gor 7 g, of cholesterol per 100 g sample. Other compositions, such as aMOPL 30 product, can include about 2-4 g, or about 3 g, of cholesterolper 100 g sample.

The compositions can also include an extremely low water content. A lowwater content can increase the stability and shelf life of thecompositions, thereby providing a high purity product. In someembodiments, the phospholipid composition will include less than about 2wt. % water, less than about 1 wt. % water, less than about 0.75 wt. %water, less than about 0.5 wt. % water, less than about 0.4 wt. % water,less than about 0.25 wt. % water, less than about 0.1 wt. % water, lessthan about 0.05 wt. % water, or less than about 0.01 wt. % water.

High levels of free fatty acids contribute to rancidity, off-taste, andincreased oxidizability of food products and supplements. Thephospholipid compositions described herein can be provided in a formcontaining very low or no free fatty acid content, due to the lownaturally occurring levels in herring roe and the gentle and mildextraction and purification steps employed. The phospholipidcompositions obtained from fish roe typically contain much lower amountof free fatty acids compared to products extracted from other sourcessuch as hill, the extracts from which can include high amounts of freefatty acids (e.g., greater than about 3 wt. %, and as high as about21-22 wt. %). The phospholipid compositions described herein can alsoinclude specific beneficial amounts of phospholipids andlysophospholipds not found in other extracts and compositions. Forexample, the phospholipid compositions described herein can include alysophospholipid (e.g., a 1-acyl; 2-lyso; 1-lyso; 2-acyl; or di-lyso(phosphatidic acid); an alkylacyl PC and/or PE; an alkenylacyl PC and/orPE; and/or other phospholipids Such components are often ignored, suchas phosphatidyl glycerol (PG) and diphosphatidylglycerol (DPG orcardiolipin), all of which are important bioactive lipids often ignoredor not found in useful quantities in extractions from fish products orby-products. The extracts can also include astaxanthin (e.g., at about1-1000 ppm).

The methods of efficiently extracting phospholipids from fishcompositions such as fish roe provide a novel composition of solidphospholipids. While other phospholipids have been extracts from variousfish compositions, the inventors have identified a unique set ofphospholipids that can provide beneficial qualities to therapies andfood products, where certain components are found in very highquantities, and other species are found in very low quantities or theyare absent. Species-specific naturally low levels of free fatty acidscombined with a gentle extraction technique have enabled the inventorsto obtain a novel and advantageous phospholipid composition as describedherein.

Thus, in some embodiments, with respect to the total mass of the PL wax,the PL wax can include at least about 50 wt. %, at least about 51 wt. %,or at least about 55 wt. % of phospholipids.

With respect to the total mass of phospholipids in the PL wax, the PLwax can include at least about 75 wt. %, at least about 80 wt. %, atleast about 81 wt. %, or at least about 83 wt. % of phosphatidylcholine.With respect to the total mass of phospholipids in the PL wax, the PLwax can include less than about 5 wt. %, less than about 4 wt. %, lessthan about 3 wt. %, less than about 2.5 wt. % of phosphatidylinositol,or substantially no phosphatidylinositol (i.e., no detectable amount).With respect to the total mass of phospholipids in the PL wax, the PLwax can include about 5-10 wt. %, or about 6-9 wt. % ofphosphatidylethanolamine. In other embodiments, the PL wax can includeless than about 10 wt. %, less than about 9 wt. %, or less than about 8wt. % of phosphatidylethanolamine. With respect to the total mass ofphospholipids in the PL wax, the ratio of phosphatidylcholine tophosphatidylethanolamine can be from about 8:1 to about 17:1.

The PL wax can also have less than about 2 wt. %, less than about 1 wt.%, or no detectable phosphatidylserine, and less than about 2 wt. %,less than about 1 wt. %, or no detectable sphingomyelin.

The invention also provides a dietary supplement, a nutritionalsupplement or a food that includes a PL wax or MOPL compositiondescribed herein, or one made by a processes described herein. Thus, theinvention provides an oral delivery vehicle, a topical delivery vehicle,a sub-lingual delivery vehicle, a parenteral delivery vehicle, or foodmade by a processes described herein, or that includes a compositionsdescribed herein.

To extract the phospholipids from the fish, fish by-products, or fishroe, any suitable and effective food grade polar solvent that is polarbased on dielectric constant will suffice to effect a good extraction ofthe polar phospholipids. In some embodiments, the polar solvent is, forexample, methanol, ethanol, butanol, or a combination thereof. In otherembodiments, the extraction is a supercritical fluid extraction with apolar entrainer and/or a supercritical fluid solvent such as carbondioxide.

The lipid compositions described herein can also include proteins oramino acids, such a protein from fish, from a fish by-product, or fromfish roe, as described herein. The extracted solid lipid compositionwill typically have little (less than about 4 wt. %, less than about 3wt. %, or less than about 0.5 wt. %) or no protein component because theproteins are poorly dissolved during the extraction process, and aretypically filtered off. However, certain protein components or aminoacids can be added to an emulsion prepared from the extractedphospholipid solid to provide a suitable and effective delivery vehiclefor the protein or amino acids.

Methods of Extraction

A phospholipid composition can be extracted from fish composition,particularly fish roe. The fish component can be frozen and then groundand dried. The dried material can then be extracted with a suitablefood-grade solvent, typically with stirring at ambient temperatures(˜23° C.). It can be advantageous to break any egg shells to improve theefficiency of the extraction process. The solids of the mixture can thenbe separated, for example by decanting or the like. It can beadvantageous to re-extract the solids at a higher concentration of thefood-grade solvent, followed by separation of the solids from theextract. The extracts can then be optionally combined. The solvent canthen be removed by any suitable and effective method. It can beadvantageous to provide heat to enhance the evaporation, although thecomposition is typically heated to only 55° C. or less.

When the extract reaches approximately 10 wt. %, 15 wt. %, or 20 wt. %lipids, non-soluble solids can be removed by filtration. The lipidcontent of the extracts can be determined by evaporation of volatilesfrom a sample using a rotary evaporator under reduced pressure, or byinfrared measurements. The solvent removal can be continued until theextract reaches about 50 wt. % to about 60 wt. % lipids, at which pointthe extract can be centrifuged to remove insoluble particles. Theremaining solvent is then removed from the extract to provide a solidwith a light amber color and a high content of phospholipids.

A phospholipid composition with an even higher content of phospholipidscan be obtained by increasing the polarity of the extraction solvent.For example, instead of using 96% ethanol, a solvent with a higherpercentage of water can be used, in order to extract a higher percentageof polar phospholipids, and thereby reducing the extraction of lesspolar components (e.g., certain fats and cholesterol).

Supplements and Food Products

The polar lipid compositions described herein can be used as anutritional supplement, or it can be added to a food to provide afunctional food. The phospholipid composition can be processed into apowder or granulated for such uses. Suitable methods include freezedrying the polar lipid composition in the presence of a suitableexcipient. The resulting powder or granulated formulation can then beincorporated into oral delivery vehicles, dietary supplements,nutritional supplements, cosmetics, cosmeceuticals, and food productssuch as fortified foods and functional foods.

Suitable phospholipids can be obtained as described herein, such as frompelagic sources including herring roe. These extractions can be referredto as a PL was and they can be combined with a carrier oil to providecompositions referred to as Marine Omega-3 Phospholipids (MOPL). Thephospholipid products can be kosher or halal when proper processingguidelines are followed, and they can be free of allergens such asshellfish allergens. The compositions can also advantageously be used inproducts such as infant formulas.

The invention also provides dietary supplements comprising the polarlipid compositions described above and processes for making the dietarysupplements. Other nutraceuticals agents may also be included in thesupplement. Nutraceutical agents are natural, bioactive chemicalcompounds that have health promoting, disease preventing or medicinalproperties. Examples of nutraceuticals include, but are not limited to,Allium cepa, Allium sativum, Aloe vera, Angelica Species, NaturallyOccurring Antioxidants, Aspergillus oryzae, barley grass, Bromelain,Carnitine, carotenoids and flavonoids, Catechin, Centella asiatica (Gotukola), Coenzyme Q₁₀, Chinese Prepared Medicines, Coleus forskohlii,Commiphora mukul, Conjugated Linoleic Acids (CLAs), Crataegus oxyacantha(Hawthorne), Curcuma longa (Turmeric), Echinacea Species (PurpleConeflower), Eleutherococcus senticosus (Siberian Ginseng), EphedraSpecies, Dietary Fish Oil, Genistein, Ginkgo biloba, Glycyrrhiza(Licorice), Hypericum perforatum (St. John's Wort), Hydrastis(Goldenseal) and other Berberine-containing plants, Lactobacillus,Lobelia (Indian Tobacco), Melaleuca alternifolia, Menaquinone, Menthapiperita, n-glycolylneuraminic acid (NGNA), Panax Ginseng, PancreaticEnzymes, Piper mythisticum, Procyanidolic Oligomers, Pygeum africanum,Quercetin, Rosemary/Lemon balm, Sarsaparilla species, Serenoa repens(Saw palmetto, Sabal serrulata), Silybum marianum (Milk Thistle),Selenite, Tabebuia avellanedae (LaPacho), Taraxacum officinale,Tanacetum parthenium (Feverfew), Taxol, Uva ursi (Bearberry), Vacciniummyrtillus (Blueberry), Valerian officinalis, Viscum album (Mistletoe),Vitamin E, Vitamin A, Beta-Carotene and other carotenoids, and Zingiberofficinale (Ginger).

Such compositions may contain, for example, a daily dosage of about 0.1g to about 5.0 g of the polar lipid composition. Furthermore, thedietary supplement is preferably provided in an amount sufficient toinduce the physiological response desired (e.g., treatment orprophylaxis of a condition such as high blood triglycerides, highcholesterol, inflammation, hypertension, metabolic syndrome, obesity,cognitive decline, memory loss, etc. A variety of uses is described inmore detail below.

The dietary supplements of the invention are further useful inconjunction with a weight loss diet regimen. The invention is notlimited to a particular kind of weight loss diet regimen (e.g.,exercise, reduced calorie intake, etc.). In preferred embodiments, theweight loss diet regimen is a dietary plan (e.g., Atkins diet, BeverlyHills diet, Cabbage Soup diet, DietSmart.com diet, DietWatch.com diet,Fit For Life diet, Grapefruit diet, Herbalife diet, High Protein diet,Jenny Craig diet, Juice Fasts diet, Kashi GoLean diet, Low Fat diet,Mayo Clinic diet, Nutrisystem diet, Perricone diet, Pritkin diet, Readyto Eat diet, Revival Soy diet, Richard Simmons diet, Scarsdale diet,Shakes diet, Slim-Fast diet, Somersizing diet, South Beach diet, SpecialK diet, Subway diet, Sugar Busters diet, Thin For Life diet, WeightWatchers diet, and Zone diet). In still other preferred embodiments, theweight loss diet regimen is an exercise plan (e.g., running, swimming,meditation, yoga, clinical therapy, bicycling, walking, etc.). In stillother preferred embodiments, the weight loss diet regimen is aclinically assisted plan (e.g., hypnosis, rehabilitory training, adietary plan provided through a dietician, surgical procedures, etc.).

The dietary supplements of the invention may further be administered inany form (e.g., pill, food product, etc.). In preferred embodiments, thedietary supplements are provided as a beverage, bar, powder, pill, orshake (e.g., a nutritional supplement as described in more detailbelow).

The dietary supplements of the invention may be taken one or more timesdaily. Preferably, the dietary supplement is administered orally one totwo times daily. Frequency of administration will, of course, depend onthe dose per unit (capsule or tablet) and the desired level ofingestion. Dose levels/unit can be adjusted to provide the recommendedlevels of ingredients per day (e.g., approximately 0.1-5 g of the polarlipid composition) in a reasonable number of units (e.g., two capsulesor tablets taken twice a day). In preferred embodiments, the doses addup each day to the daily intake of each ingredient. In preferredembodiments, the dietary supplements are taken with meals or beforemeals. In other embodiments, the dietary supplements are not taken withmeals.

Dietary supplements of the invention may be delivered in any suitableformat, including, but not limited to, dermal delivery, oral delivery,or mucosal delivery. The ingredients of the dietary supplement caninclude pharmaceutically acceptable excipients and/or carriers for oralconsumption, and in particular in the form of an oral delivery vehicle.The carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet(coated or non-coated), tea, or the like. The dietary supplement ispreferably in the form of a tablet or capsule and most preferably in theform of a hard gelatin capsule. Suitable excipient and/or carriersinclude maltodextrin, calcium carbonate, dicalcium phosphate, tricalciumphosphate, microcrystalline cellulose, dextrose, rice flour, magnesiumstearate, stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). Preferred carriers include calcium carbonate,magnesium stearate, maltodextrin, and mixtures thereof. The variousingredients and the excipient and/or carrier are mixed and formed intothe desired form using conventional techniques. The tablet or capsule ofthe invention may be coated with an enteric coating that dissolves at apH of about 6.0 to 7.0. A suitable enteric coating that dissolves in thesmall intestine but not in the stomach is cellulose acetate phthalate.Further details on techniques for formulation for and administration maybe found in Remington: The Science and Practice of Pharmacy, 21^(st) Ed.(Lippincott Williams & Wilkins, Philadelphia, Pa.; 2005).

In other embodiments, the supplement is provided as a powder or liquidsuitable for adding by the consumer to a food or beverage. For example,in some embodiments, the dietary supplement can be administered to anindividual in the form of a powder, for instance to be used by mixinginto a beverage, or by stirring into a semi-solid food such as apudding, topping, sauce, puree, cooked cereal, or salad dressing, forinstance, or by otherwise adding to a food.

The dietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementof the invention may also contain optional ingredients including, forexample, herbs, vitamins, minerals, enhancers, colorants, sweeteners,flavorants, inert ingredients, and the like. For example, the dietarysupplement of the invention may contain one or more of the following:asorbates (ascorbic acid, mineral ascorbate salts, rose hips, acerola,and the like), dehydroepiandosterone (DHEA), Fo-Ti or Ho Shu Wu (herbcommon to traditional Asian treatments), Cat's Claw (ancient herbalingredient), green tea (polyphenols), inositol, kelp, dulse,bioflavinoids, maltodextrin, nettles, niacin, niacinamide, rosemary,selenium, silica (silicon dioxide, silica gel, horsetail, shavegrass,and the like), spirulina, zinc, and the like. Such optional ingredientsmay be either naturally occurring or concentrated forms.

In some embodiments, the dietary supplements further comprise vitaminsand minerals including, but not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D₃; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.Suitable dosages for vitamins and minerals may be obtained, for example,by consulting the U.S. RDA guidelines.

In other embodiments, the invention provides nutritional supplements(e.g., energy bars or meal replacement bars or beverages) comprising thepolar lipid compositions described above. The nutritional supplement mayserve as meal or snack replacement and generally provide nutrientcalories. Preferably, the nutritional supplements provide carbohydrates,proteins, and fats in balanced amounts. The nutritional supplement canfurther comprise carbohydrate, simple, medium chain length, orpolysaccharides, or a combination thereof. A simple sugar can be chosenfor desirable organoleptic properties. Uncooked cornstarch is oneexample of a complex carbohydrate. If it is desired that it shouldmaintain its high molecular weight structure, it should be included onlyin food formulations or portions thereof which are not cooked or heatprocessed since the heat will break down the complex carbohydrate intosimple carbohydrates, wherein simple carbohydrates are mono- ordisaccharides. The nutritional supplement contains, in one embodiment,combinations of sources of carbohydrate of three levels of chain length(simple, medium and complex; e.g., sucrose, maltodextrins, and uncookedcornstarch).

Sources of protein to be incorporated into the nutritional supplement ofthe invention can be any suitable protein utilized in nutritionalformulations and can include whey protein, whey protein concentrate,whey powder, egg, soy flour, soy milk soy protein, soy protein isolate,caseinate (e.g., sodium caseinate, sodium calcium caseinate, calciumcaseinate, potassium caseinate), animal and vegetable protein andmixtures thereof. When choosing a protein source, the biological valueof the protein should be considered first, with the highest biologicalvalues being found in caseinate, whey, lactalbumin, egg albumin andwhole egg proteins. In a preferred embodiment, the protein is acombination of whey protein concentrate and calcium caseinate. Theseproteins have high biological value; that is, they have a highproportion of the essential amino acids. See Modern Nutrition in Healthand Disease, eighth edition, Lea and Febiger, publishers, 1986,especially Volume 1, pages 30-32.

The nutritional supplement can also contain other ingredients, such asone or a combination of other vitamins, minerals, antioxidants, fiberand other dietary supplements (e.g., protein, amino acids, choline,lecithin, omega-3 fatty acids). Selection of one or several of theseingredients is a matter of formulation, design, consumer preference andend-user. The amounts of these ingredients added to the dietarysupplements of this invention are readily known to the skilled artisan.Guidance to such amounts can be provided by the U.S. RDA doses forchildren and adults. Further vitamins and minerals that can be addedinclude, but are not limited to, calcium phosphate or acetate, tribasic;potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodiumchloride); potassium chloride or acetate; ascorbic acid; ferricorthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D₃; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.

Flavors, coloring agents, spices, nuts and the like can be incorporatedinto the product. Flavorings can be in the form of flavored extracts,volatile oils, chocolate flavorings, peanut butter flavoring, cookiecrumbs, crisp rice, vanilla or any commercially available flavoring.Examples of useful flavoring include, but are not limited to, pure aniseextract, imitation banana extract, imitation cherry extract, chocolateextract, pure lemon extract, pure orange extract, pure peppermintextract, imitation pineapple extract, imitation rum extract, imitationstrawberry extract, or pure vanilla extract; or volatile oils, such asbalm oil, bay oil, bergamot oil, cedarwood oil, walnut oil, cherry oil,cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolateflavoring, vanilla cookie crumb, butterscotch or toffee. In oneembodiment, the dietary supplement contains cocoa or chocolate.

Emulsifiers may be added for stability of the final product. Examples ofsuitable emulsifiers include, but are not limited to, lecithin (e.g.,from egg or soy), and/or mono- and di-glycerides. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

Preservatives may also be added to the nutritional supplement to extendproduct shelf life. Preferably, preservatives such as potassium sorbate,sodium sorbate, potassium benzoate, sodium benzoate or calcium disodiumEDTA are used.

In addition to the carbohydrates described above, the nutritionalsupplement can contain natural or artificial (preferably low calorie)sweeteners, e.g., saccharides, cyclamates, aspartamine, aspartame,acesulfame K, and/or sorbitol. Such artificial sweeteners can bedesirable if the nutritional supplement is intended to be consumed by anoverweight or obese individual, or an individual with type II diabeteswho is prone to hyperglycemia.

The nutritional supplement can be provided in a variety of forms, and bya variety of production methods. In a preferred embodiment, tomanufacture a food bar, the liquid ingredients are cooked; the dryingredients are added with the liquid ingredients in a mixer and mixeduntil the dough phase is reached; the dough is put into an extruder, andextruded; the extruded dough is cut into appropriate lengths; and theproduct is cooled. The bars may contain other nutrients and fillers toenhance taste, in addition to the ingredients specifically listedherein.

Servings of the nutritional supplement preferably contain for example, adaily dosage of between 0.1 g and 5.0 g of the polar lipid composition.It is understood by those of skill in the art that other ingredients canbe added to those described herein, for example, fillers, emulsifiers,preservatives, etc. for the processing or manufacture of a nutritionalsupplement.

In still further embodiments, the invention provides functional foods,including food products, prepared food products, or foodstuffscomprising the polar lipid compositions described above. For example, insome embodiments, beverages and solid or semi-solid foods comprising thepolar lipid compositions (or a powder or granulated formulation thereof)are provided. These forms can include, but are not limited to, beverages(e.g., soft drinks, milk and other dairy drinks, and diet drinks), bakedgoods, puddings, dairy products, confections, snack foods, or frozenconfections or novelties (e.g., ice cream, milk shakes), prepared frozenmeals, candy, snack products (e.g., chips), soups, spreads, sauces,salad dressings, prepared meat products, cheese, yogurt and any otherfat or oil containing foods, and food ingredients (e.g., wheat flour).Servings of the food product preferably contain between 0.1 g and 5.0 gof the polar lipid composition.

Many important nutraceutical or cosmeceutical ingredients are poorlysoluble in both aqueous compositions and lipophilic compositions. Suchingredients are also often poorly absorbed by the body. Examples includeisoflavonoids and quercetin. Adding poorly soluble components to a MOPLcomposition or to an emulsion prepared from a PL wax or MOPL compositioncan improve their solubility, absorbability, and bio-efficacy. Thenutraceutical or cosmeceutical ingredients can also be added to aphospholipid fraction extracted from fish composition, such as herringroe, prior to the removal of the polar solvent. The mixture can then beformulated into an emulsion or MOPL composition for providing improvedsolubility, absorbability, and bio-efficacy, as well as a high omega-3content.

Pharmaceutical Formulations

The phospholipid compositions described herein can be used to preparetherapeutic pharmaceutical compositions. The phospholipid compositionsare dispersible in water and oil, therefore they are amenable to a widerange of applications. Thus, the compositions described herein can beformulated as pharmaceutical compositions and administered to amammalian host, such as a human patient, in a variety of forms. Theforms can be specifically adapted to a chosen route of administration,e.g., topical, oral, or parenteral administration, such as byintravenous, intramuscular, or subcutaneous routes.

The compositions described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compounds can be enclosed in hard or soft shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compositions may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such formulations and preparations typically contain at least 0.1% ofthe phospholipid composition. The percentage of phospholipid in thecompositions and preparations can vary and may conveniently be about 1%to about 99%, about 2% to about 90%, or about 2% to about 60% of theweight of a given unit dosage form. The amount of active in suchtherapeutically useful compositions is such that an effective dosagelevel can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, thephospholipids may be incorporated into sustained-release preparationsand devices.

In various embodiments, the phospholipids can be administeredintravenously or intraperitoneally by infusion or injection. Dispersionsof the phospholipids can be prepared in water, optionally mixed with anontoxic surfactant. Solutions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain one or more antioxidants orpreservatives, for example, to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active phospholipids adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and/or suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thiomersal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating thephospholipids in an appropriate amount in a suitable solvent, optionallywith various other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation caninclude vacuum drying and freeze drying techniques, which yield a powderof the ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

Topical Formulations.

For topical administration, phospholipids may be applied in pure form,e.g., as a PL wax or MOPL oil. However, it will generally be desirableto administer the phospholipids to the skin as a composition orformulation, for example, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which the phospholipids can be dissolvedor dispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Topical treatments, for example, in the form of ointments and creams,can be based on a composition described herein in combination withcortisone and/or vitamin D derivatives. Retinoids (vitamin A derivative)may also be used, alone or in combination with the with cortisone and/orvitamin D derivatives. These combinations are useful, for example, fortreating psoriasis conditions such as small psoriasis lesions, or fortreating a slow progression of psoriasis.

Examples of dermatological compositions for delivering active agents tothe skin are known to the art; for example, see U.S. Pat. Nos. 4,992,478(Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157(Smith et al.). Such dermatological ingredients and compositions can beused in combinations with the phospholipids described herein.

Formulation Dosage.

Useful dosages of the compositions described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a composition requiredfor use in treatment will vary not only with the particular set ofphospholipids selected but also with the route of administration, thenature of the condition being treated, and the age and condition of thepatient, and will be ultimately at the discretion of an attendantphysician or clinician.

In some embodiments, the compound can be conveniently administered in aunit dosage form, for example, containing about 5 to 1000 mg/m², about10 to 750 mg/m², or about 50 to 500 mg/m² of phospholipid ingredient perunit dosage form. The desired dose may conveniently be presented in asingle dose or as divided doses administered at appropriate intervals,for example, as two, three, four or more sub-doses per day. The sub-doseitself may be further divided, e.g., into a number of discrete looselyspaced administrations.

Alternatively, an effective dosage amount of a phospholipid compositiondescribed herein can include, for example, about 300 mg to about 1000 mgof total omega-3 fatty acids. In various embodiments, the phospholipidcomposition can be administered orally. In other embodiments, thephospholipid composition can be administered topically. In someembodiments, the phospholipid composition is provided in a gel capsuleor pill.

Therapy for Diseases and Adverse Conditions

Omega-3 fatty acids, in particular EPA and DHA, play a vital role incentral nervous system, cognitive, cardiovascular, joint, immune, andmetabolic functions. EPA and DHA not only protect good overall physicaland emotional health, but also can reduce the risk of cardiac diseaseand exert powerful anti-inflammatory effects that can help treat certaindiseases. The benefits of EPA and DHA have been studied across a widerange of illnesses, including, but not limited to, heart disease, highcholesterol, hypertension, arthritis, back pain, osteoporosis,psoriasis, lupus, Crohn's Disease, back pain, dry eyes, depression,bipolar disorder, ADHD, and stress-related disorders. Omega-3 fattyacids have also been shown to be important in pregnant women andinfants, where their depletion can lead to visual or central nervoussystem problems. Additionally, omega-3 fatty acids are important forproper functioning in companion and husbandry animals. However, mostsources of nutritional fats do not have a healthy ratio of omega-3 toomega-6 fatty acids. They also contain only low amounts of importantomega-3 fatty acids such as DHA and EPA, and they are not highlybioavailable from those sources. The polar lipid compositions describedherein can be used to provide concentrated amounts of omega-3 fattyacids that are highly bioavailable, which can aid the therapeutictreatment of many conditions, such as those described below.

Accordingly, the polar lipid compositions described herein have a widevariety of uses. The phospholipids have anti-inflammatory properties andhave shown to be beneficial for brain health and cognitive performance.The phospholipid compositions are also beneficial for maintaining andimproving gut health and cardiovascular health. For example, the polarlipid compositions are useful in modulating plasma triglyceride levelsas well as plasma HDL C levels (the amount of cholesterol contained inHDL particles), while not elevating LDL C levels. Relevant diseases anddisorders that can be treated include but are not limited tocardiometabolic disorders/metabolic syndrome (MetS), neurodevelopmentaland neurodegenerative diseases/disorders, and inflammation disorders.

The invention thus provides methods of treating or preventing acardiometabolic disorder/metabolic syndrome is provided, the methodcomprising administering to a subject in need thereof a polar lipidcomposition as described above. In some embodiments, the cardiometabolicdisorder can be atherosclerosis, arteriosclerosis, coronary heart(carotid artery) disease (CHD or CAD), acute coronary syndrome (or ACS),valvular heart disease, aortic and mitral valve disorders,arrhythmia/atrial fibrillation, cardiomyopathy and heart failure, anginapectoris, acute myocardial infarction (or AMI), hypertension,orthostatic hypotension, shock, embolism (pulmonary and venous),endocarditis, diseases of arteries, the aorta and its branches,disorders of the peripheral vascular system (peripheral arterial diseaseor PAD), Kawasaki disease, congenital heart disease (cardiovasculardefects) and stroke (cerebrovascular disease), dyslipidemia,hypertriglyceridemia, hypertension, heart failure, cardiac arrhythmias,low HDL levels, high LDL levels, stable angina, coronary heart disease,acute myocardial infarction, secondary prevention of myocardialinfarction, cardiomyopathy, endocarditis, type 2 diabetes, insulinresistance, impaired glucose tolerance, hypercholesterolemia, stroke,hyperlipidemia, hyperlipoproteinemia, chronic kidney disease,intermittent claudication, hyperphosphatemia, omega-3 deficiency,phospholipid deficiency, carotid atherosclerosis, peripheral arterialdisease, diabetic nephropathy, hypercholesterolemia in HIV infection,non-alcoholic fatty liver disease/non-alcoholic steatohepatitis(NAFLD/NASH), arterial occlusive diseases, cerebral atherosclerosis,arteriosclerosis, cerebrovascular disorders, myocardial ischemia,coagulopathies leading to thrombus formation in a vessel, or diabeticautonomic neuropathy.

Methods of treating, preventing, or improving cognition and/or acognitive disease, disorder or impairment (memory, concentration,learning (deficit)), or of treating or preventing neurodegenerativedisorders are also provided. The methods can include administering to asubject in need thereof a polar lipid composition as described herein.In some embodiments, the cognitive disease, disorder or impairment isAttention Deficit Disorder (ADD), Attention Deficit HyperactivityDisorder (ADHD), autism/autism spectrum disorder (ASD), dyslexia,age-associated memory impairment and learning disorders, amnesia, mildcognitive impairment, cognitively impaired non-demented, pre-Alzheimer'sdisease, Alzheimer's disease, epilepsy, Pick's disease, Huntington'sdisease, Parkinson's disease, Lou Gehrig's disease, pre-dementiasyndrome, Lewy body dementia dementia, dentatorubropallidoluysianatrophy, Freidreich's ataxia, multiple system atrophy, types 1, 2, 3, 6,7 spinocerebellar ataxia, amyotrophic lateral sclerosis, familialspastic paraparesis, spinal muscular atrophy, spinal and bulbar muscularatrophy, age-related cognitive decline, cognitive deterioration,moderate mental impairment, mental deterioration as a result of ageing,conditions that influence the intensity of brain waves and/or brainglucose utilization, stress, anxiety, concentration and attentionimpairment, mood deterioration, general cognitive and mental well-being,neurodevelopmental, neurodegenerative disorders, hormonal disorders,neurological imbalance, or a combinations thereof. In a specificembodiment, the cognitive disorder is memory impairment. In someinstances, the methods described above for treating, preventing, orimproving cognition and/or a cognitive disease, disorder or impairment(memory, concentration, learning (deficit)), or of treating orpreventing neurodegenerative disorders may utilize the polar lipidcompositions described herein.

In some embodiments, methods for inhibiting, preventing, or treatinginflammation or an inflammatory disease are provided, the methodscomprising administering to a subject in need thereof, a polar lipidcomposition as described above. In some embodiments, the inflammation orinflammatory disease is selected from organ transplant rejection;reoxygenation injury resulting from organ transplantation (see Grupp etal., J. Mol. Cell. Cardiol. 31: 297-303 (1999)) including, but notlimited to, transplantation of the following organs: heart, lung, liverand kidney; chronic inflammatory diseases of the joints, includingarthritis, rheumatoid arthritis, osteoarthritis and bone diseasesassociated with increased bone resorption; inflammatory bowel diseases(IBD) such as ileitis, ulcerative colitis (UC), Barrett's syndrome, andCrohn's disease (CD); inflammatory lung diseases such as asthma, acuterespiratory distress syndrome (ARDS), and chronic obstructive pulmonarydisease (COPD); inflammatory diseases of the eye including cornealdystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmitisand endophthalmitis; chronic inflammatory diseases of the gum, includinggingivitis and periodontitis; inflammatory diseases of the kidneyincluding uremic complications, glomerulonephritis and nephrosis;inflammatory diseases of the skin including sclerodermatitis, psoriasisand eczema; inflammatory diseases of the central nervous system,including chronic demyelinating diseases of the nervous system, multiplesclerosis, AIDS-related neurodegeneration and Alzheimer's disease,infectious meningitis, encephalomyelitis, Parkinson's disease,Huntington's disease, Epilepsy, amyotrophic lateral sclerosis and viralor autoimmune encephalitis, preeclampsia; chronic liver failure, brainand spinal cord trauma, or cancer. The inflammatory disease can also bea systemic inflammation of the body, exemplified by gram-positive orgram negative shock, hemorrhagic or anaphylactic shock, or shock inducedby cancer chemotherapy in response to proinflammatory cytokines, e.g.,shock associated with proinflammatory cytokines. Such shock can beinduced, e.g., by a chemotherapeutic agent that is administered as atreatment for cancer. Other disorders that can be treated includedepression, obesity, allergic diseases, acute cardiovascular events,muscle wasting diseases, and cancer cachexia. Also, inflammation thatresults from surgery and trauma can be treated with the polar lipidcompositions.

In some embodiments, the invention provides methods for reducingsymptoms of cognitive dysfunction in a child. The methods can includeadministering an effective amount of a phospholipid compositiondescribed herein to a child in need of such symptom reduction. Thesymptoms can be, for example, one or more of the ability to completetask, ability to stay on task, ability to follow instructions, orability to complete assignments. Administration of a compositiondescribed herein can improve psychomotor function, long term memory,short term memory, ability to make a decision, ability to follow throughon decision, ability to self-sustain attention, ability to engage inconversations, sensitivity to surroundings, ability to plan, ability tocarry out plan, ability to listen, interruptions in social situations,temper tantrums, level/frequency of frustration, level/frequencyrestlessness, frequency/level fidgeting, ability to exhibit delayedgratification, aggressiveness, demanding behavior/frequency of demandingbehavior, sleep patterns, restive sleep, interrupted sleep, awakeningbehavior, disruptive behavior, ability to exhibit control in socialsituations, ability to extrapolate information and/or ability tointegrate information. In some embodiments, the subject can exhibit oneor more symptoms of Attention Deficit Hyperactivity Disorder (ADHD), canbe suspected of having ADHD, or can have been diagnosed with ADHD. Insome embodiments, the subject exhibits one or more symptoms of autisticspectrum disorder, is suspected of having autistic spectrum disorder, orhas been diagnosed with autistic spectrum disorder.

In further embodiments, the invention provides methods of increasingcognitive performance in an aging mammal. The methods can includeadministering an effective amount of a phospholipid compositiondescribed herein. In some embodiments, the cognitive performance ismemory loss, forgetfulness, short-term memory loss, aphasia,disorientation, disinhibition, or undesired behavioral changes.

The invention also provides methods of treating a subject byadministration of a phospholipid composition described herein to asubject under conditions such that a desired condition is improved. Thecondition can be, for example, fertility, physical endurance, sportsperformance, muscle soreness, inflammation, auto-immune stimulation,metabolic syndrome, obesity, or type II diabetes.

In some embodiments, the invention provides methods for prophylacticallytreating a subject by administration of a phospholipid compositiondescribed herein to a subject under conditions such that an undesirablecondition is prevented. The undesirable condition can be, for example,weight gain, infertility, obesity, metabolic syndrome, diabetes type II,mortality in subjects with a high risk of sudden cardiac death, orinduction of sustained ventricular tachycardia. In some embodiments, thesubject is at risk for developing a condition of weight gain, obesity,metabolic syndrome, diabetes type II, mortality in subjects with a highrisk of sudden cardiac death, or induction of sustained ventriculartachycardia.

The compositions described herein can also be used for treating skinconditions such as common psoriasis, guttate psoriasis, nummularpsoriasis, plaque psoriasis, erythrodermic psoriasis, psoriaticarthritis, pustular psoriasis, child psoriasis, parapsoriasis, acute orchronic dermatitis such as ichtiosis and keratose dermatitis, includingpalmoplantar keratoderma.

The subject or patient treated by the methods described herein can be amammal. A mammal includes a primate, human, rodent, canine, feline,bovine, ovine, equine, swine, caprine, bovine and the like. In someembodiments, the mammal is a human. In some embodiments, the human is amale; in other embodiments, the human is a female. In certainembodiments, the subject is a companion animal. In yet otherembodiments, the mammal is a pet such as a cat or dog. In furtherembodiments, the mammal has symptoms of age-associated memory impairmentor decline.

The invention also provides phospholipid compositions that can beformulated into a feed product. Such feed products can reduce low-gradechronic inflammation in animals. The phospholipid compositions can alsobe formulated into a food product and given to humans for the samepurpose. Furthermore, the phospholipid compositions can be formulated asa functional food product, as a drug or as food supplement.

Herring roe PL wax and MOPL are novel and convenient sources of EPA.They are also particularly good sources of DHA for infants. In addition,the phospholipid compositions described herein contain a highlyavailable source of cholesterol for infants. Cholesterol is a keynutrient for infants and is lacking in vegetable oil-based infantformulas. Studies with sows demonstrated that the cholesterol in sowmilk was much more bioavailable in the milk fat globule membrane (MFGM)than when added to infant formula. Therefore, the phospholipids in MOPLcan make the cholesterol more bioavailable to those in need ofcholesterol, such as infants. In addition, the phospholipid compositionsalso provide high bioavailability of other important dietary componentssuch as choline.

The invention also provides therapeutic methods of treating cancer in amammal. The methods can include administering to a mammal having canceran effective amount of a composition described herein. Cancer refers toany various type of malignant neoplasm, for example, colon cancer,breast cancer, melanoma and leukemia, and in general is characterized byan undesirable cellular proliferation, e.g., unregulated growth, lack ofdifferentiation, local tissue invasion, and metastasis. The ability of acompound of the invention to treat cancer may be determined by usingassays well known to the art. For example, the design of treatmentprotocols, toxicity evaluation, data analysis, quantification of tumorcell kill, and the biological significance of the use of transplantabletumor screens are known.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

Example 1 Extraction of Phospholipids

Frozen, immature herring roe was ground and vacuum dried. The driedmaterial was extracted with 96% ethanol at a ratio of 10 liters ofethanol per kg of dried roe in a stirred reactor at ambient temperaturefor 30 minutes. The stifling inside the reactor was combined with anexternal loop via a Silverson high shear mixer to break egg shellsduring the extraction. The solids and liquid extract were separated witha decanter. The solids were re-extracted with 96% ethanol, approximately6 liters of ethanol per kg of dry weight eggs, for another 30 minutes.The solids and liquids were again separated in a decanter. The combinedliquids (extracts) had a lipid content of around 5 wt. %, as determinedby evaporation of volatiles from a sample using a rotary evaporatorunder reduced pressure. The lipid fraction was then concentrated byethanol evaporation until reaching approximately 12% in a falling filmevaporator at a temperature of less than 50° C. The lipid fraction wasfurther concentrated by ethanol removal under reduced pressure in astirred reactor. At a lipid content of around 20%, the ethanol solutionwas filtered to remove non-soluble solids. The concentration thencontinued until reaching approximately the 55% level at a temperature ofless than 55° C. The 55% extract can then optionally be centrifuged toremove insoluble particles. The composition was then subjected to afinal evaporation in a Gueudu mixer under reduced pressure. Pure ethanolcan optionally be added and evaporated to assist the evaporation offinal amounts of water to below 1%. The resulting product was a solidwith a light amber color and a high content of phospholipids.

Example 2 Analysis of Phospholipid Composition Content

Extracted phospholipid compositions were in the form of a solid PL wax,which were analyzed for phospholipid mass balance and fatty acidcontent. The phospholipid compositions can be about 50% to about 95%phospholipids by weight. With further purification, the compositions canbe about 90 wt. % to about 100 wt. % phospholipids. As can be observedfrom Table 2-1 below, phosphatidylcholine forms a large percentage ofthe phospholipid composition (e.g., a PL wax or a PL oil), for example,at least about 40 wt. %, at least about 50 wt. %, or at least about 54wt. %. The composition also includes at least about 5 wt. %, or at leastabout 6 wt. % phosphatidylethanolamine. Table 2-1 uses standardabbreviations including APE for acyl phosphatidylethanolamine.

TABLE 2-1 Typical PL species as quantified by NMR. PL-class No of FAs MWTypical weight % of PL wax PC 2 812 55.0 1-LPC 1 534.5 0.4 2-LPC 1 534.52.7 PI 2 907 0.8 PE 2 770 6.5 LPE 1 492.5 0.4 APE 3 1032 0.3 other 2 8120.2 Sum 66

In some embodiments, the approximate mass balance of the composition canbe as shown in Table 2-2 below.

TABLE 2-2 Approximate PL Wax Total Mass Balance. Fatty acids as mg TAG/g61 Polar groups of PLs 19 Cholesterol 7 Ash content 4 Protein 3 Ethanol1 Water 0.5 Other 4.5 Sum 100

As shown in Table 2-3 below, the phospholipid compositions obtained bythe methods described in Example 1 provide a composition with a highDHA:EPA ratio and low seasonal variability.

TABLE 2-3 Phospholipid Wax Phospholipid Composition Profile.Conventional Parameter 18:12 Fish Oil Krill Oil PL Wax Analysis Contentof ω-3 and phospholipids: Total omega-3 as % of 30% 30-35%  42% PL50includes the highest fatty acids omega-3 content available in currentproducts DHA:EPA ratio 0.7 ~0.6 2.7 PL50 includes an extremely high DHAcontent Seasonal variability in EPA High High Low Lowest seasonablevariability and DHA of products analyzed Contains MOPLs? Trace Yes YesPL50 is a good source of MOPL and lecithin Phospholipid content Trace40-45% >50% PL50 has an extremely high content of phospholipids

The phospholipid compositions have demonstrated excellent oxidativestability (with no addition of antioxidants) and they do not generatebelching with fish aromas that is often associated with orally deliveredconventional 18:12 fish oil (a common consumer complaint). The stabilityis not only superior to 18:12 fish oil but is also significantlysuperior to hill oil, which also has an inherently fishy odor that ismuch less noticeable in the phospholipid compositions described herein.Trace metals are not reported to concentrate in the roe, and there areno food chain or ecosystem concerns because the roe is a byproduct ofexisting fishery operations.

The phospholipid compositions can include a very low amount of freefatty acids (less than about 6.5 wt. %, less than about 5.5 wt. %, orless than about 1 wt. %), while krill oil can have as high as 21-22% ofits EPA and DHA in free fatty acid form, which negatively affectsbioavailability and/or leads to stability, oxidation, and sensorialproblems.

Other phospholipid compositions, such as those described by U.S. Pat.No. 7,759,325 (Dupont), provide compositions that include lecithin at10-50 wt. %, typically about 20 wt. %, of the composition. Of the totalphospholipids, such compositions include 10-75% of phosphatidylcholine,10-30% phosphatidyl inositol, 5-30% phosphatidylethanolamine, 5-20%phosphatidylserine, and 5-30% sphingomyelin, by weight. In variousembodiments, the phospholipid compositions described herein do notinclude phosphatidylserine or sphingomyelin, or they are included inonly very low amounts, and they include total phospholipid amounts ofgreater than 50% by weight.

A detailed analysis of fatty acids in a phospholipid extract (wax)provided the data shown in Table 2-4 below.

TABLE 2-4 Analysis of Fatty Acid Content. Fatty acid Name A % mg/g wax14:0 Myristic 3.5 21.3 15:0 pentadecanoic 0.5 3.0 15:1 pentadecenoic 0.10.6 16:0 Palmitic 19.2 116.9 16:1 palmitoleic 5.6 34.1 16:2hexadecadienoic 0.2 1.2 17:0 heptadecanoic 0.2 1.2 17:1 heptadecaenoic0.3 1.8 16:4 hexadecatetraenoic 0.1 0.6 18:0 Stearic 1.4 8.5 18:1 n-9Oleic 8.4 51.2 18:1 n-7 cis-vaccenic 3.2 19.5 18:2 n-6 Linoleic 1.0 6.118:3 n-6 γ-linolenic 0.1 0.6 18:3 n-3 α-linolenic 0.7 4.3 18:4 n-3Stearidonic 1.2 7.3 20:1 eicosenoic 1.8 11.0 20:2 n-6 eicosadienoic (n6)0.1 0.6 (dihomolinoleic) 20:4 n-6 Arachidonic 0.5 3.0 20:3 n-3eicosatrienoic (n3 ) 0.1 0.6 (dihomolinolenic) 20:4 n-3 eicosatetraenoic(n3) 0.6 3.7 20:5 n-3 EPA 12.8 77.5 22:1 n-11 cetoleic 0.5 3.0 22:1 n-9erucic 0.1 0.6 21:5 n-3 heneicosapentaenoic 0.2 1.2 22:4 n-6docosatetraenoic (n6) 0.2 1.2 (adrenic) 22:5 n-6 DPA n-6 0.2 1.2 22:5n-3 DPA n-3 1.0 6.1 22:6 n-3 DHA 31.3 191.8 24:1 tetracosenoic 0.6 3.7minor comp minor sum 4.3 26.2 Sum fatty acids 100.0 609.7 Totalsaturates 24.8 151.0 Total monoenes 20.6 125.5 Total n-3 47.7 291.2Total n-6 2.1 12.8 n-3/n-6 22.7

Further analyses of various phospholipid waxes provided the data ofTable 2-5.

TABLE 2-5 Approximate Fatty Acid Content in Phospholipid Waxes. In thephospholipids In the wax Fatty (approx. 65% of wax) (“polar lipidcomposition”) acid(s) Relative fatty acid composition (A %) Relativefatty acid composition (A %) Weight % EPA  6-20  6-20  4-12 DHA 20-4020-40 12-24 EPA + DHA 26-60 35-50 16-36 Total n-3 31-65 35-55 20-37DHA:EPA 1.5-3.5 1.5-3.5 1.5-3.5

Thus, 1 g of wax will contain a total amount of fatty acidscorresponding to around 610 mg of triglycerides. Another 190 mg of theremaining 390 mg can be accounted for by the polar moieties of thephospholipid molecules. Some variations from the actual content are aresult of the analytical methods employed. Cholesterol also accounts forabout 7 to about 8 wt. % of the total lipids in the wax obtained.Ethanol can comprise about 2 wt. % or less and water can be present inabout 0.5 wt. % or less.

Example 3 Phospholipid Composition PL30 and PL50

The phospholipid compositions obtained as descried in Example 1 can beconveniently blended with a carrier such as a fish oil, for example,having a 17:54 EPA:DHA ratio, to provide a MOPL. A blend containing aminimum of 30 wt. % polar lipids is herein referred to as PL30,typically made by mixing wax and fish oil carrier approximately 50:50. Ablend containing 50 wt. % polar lipids is herein referred to as PL50,typically made by mixing wax and fish oil carrier approximately 80:20.Ash content, primarily minerals, can comprise about 4-5 wt. % of thecomposition.

The PL30 and PL50 lipid compositions were further analyzed to determinetheir density, viscosity, flash points, and pour points. The dataobtained is shown in the table below.

Test PL30 PL50 Density @ 20° C. (g/cm³) 0.98 1.01 Density @ 50° C.(g/cm³) 0.96 0.98 Kinematic Viscosity @ 20° C. (mm²/s) 111.9 * KinematicViscosity @ 50° C. (mm²/s) 33.29 * Flash Point, PMCC (° C.) 130.0 55Pour Point (° C.) −21 −18 * Viscosity was greater than instrumentanalytical limits.

Example 4 Phospholipid Composition MOPL 50

A Marine Omega-3 Phospholipid (MOPL) composition was obtained fromherring roe as described in Example 1 and was combined with fish oiltriglycerides to optimize viscosity. Mixed tocopherols (300 ppm) wereintroduced with the fish oil as a natural food grade antioxidant. An80:20 mixture of the extract (PL wax) and the fish oil triglycerides(carrier), respectively, will typically provide a MOPL 50 composition(>50% polar lipids).

An analysis of a set of phospholipids (a PL wax) obtained according tothe methods of Example 1 also provided the data shown in Table 4-1(where PC=phosphatidylcholine; 1-LPC=1-lysophosphatidylcholine;2-LPC=2-lysophosphatidylcholine; PI=phosphatidylinositol;PE=phosphatidylethanolamine; LPE=lysophosphatidylethanolamine; andPS=phosphatidylserine), where the composition includes a fish oilcarrier to provide PL50.

TABLE 4-1 PL Composition Analysis for a MOPL (PL50) Containing a FishOil Carrier. Spec for U041/ PL50 lots PL50 008/Al2 ARC32414-1 ARC32414-2Total PL amount min 50   51.4 58.1 55.1 PL groups (% w/w of product) PC42.8 46.8 44.4 1-LPC 0.69 0.2 0.17 2-LPC 2.55 1.6 1.51 Total PC min 37.546.0 48.5 46.08 PI nd 1.3 1.24 PE 4.5 3.9 3.54 LPE 0.3 nd nd Total PE4.8 3.9 3.54 PS nd nd nd Sphingomyelin nd nd nd PL groups (% w/w oftotal PL) PC 83.2 80.5 80.6 1-LPC 1.3 0.3 0.3 2-LPC 5.0 2.7 2.7 Total PC89.5 83.5 83.6 PI nd 2.2 2.3 PE 8.7 6.7 6.4 LPE 0.6 nd nd Total PE 9.46.7 6.4 PS nd nd nd Sphingomyelin nd nd nd nd = not detected.

The product specification is in compliance with the EC regulations forfood, and the GOED voluntary monograph regarding safety requirements(for environmental pollutants such as dioxins, PCB, and heavy metals).When stored in unopened closed containers under recommended storageconditions, the product has a shelf life of at least 36 months.

MOPL 50 Analytical Specifications:

Parameter Unit Min. value Max. value Appearance Amber liquid oilSolubility Oil soluble and water dispersible EPA (C20:5 n-3) Area % 10DHA (C22:6 n-3) Area % 27 Total omega-3¹⁾ Area % 42 EPA (C20:5 n-3) mg/g70 DHA (C22:6 n-3) mg/g 190 Total omega-3¹⁾ mg/g 290 Total PC mg/g 375Total PL mg/g 500 Total Neutral lipids mg/g 500 Water content by KarlFisher % 1.0 Peroxide value meq/kg 3.0 Total plate count cfu/g <100 E.coli  /1 g negative Salmonella /25 g negative Total Coliforms cfu/g <10Yeast cfu/g <10 Molds cfu/g <40 Enterobacteria negative/1 g negativeStaphylococci coagulase positive negative/1 g negative Dioxins andfurans (PCDDs, PCDFs) pg WHO-TEQ/g 2.0 Dioxine-like PCBs pg WHO-TEQ/g3.0 Dioxins + furans + Dioxine-like PCBs pg WHO-TEQ/g 3.0 Benzo(a)pyreneng/g 2.0 Sum of benzo(a)pyrene, benz(a)anthracene, ng/g 10.0benzo(b)fluoranthene and chrysene PCBs IUPAC no. 28, 52, 101, 118, 138,153, 180 mg/kg 0.09 Pb, Lead mg/kg 0.1 Hg, Mercury mg/kg 0.01 As,Arsenic mg/kg 0.1 Cd, Cadmium mg/kg 0.1 ¹⁾Total n-3: EPA, DHA, 18:3,18:4, 20:4, 21:5, 22:5

Example 5 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a phospholipid composition described herein or aphospholipid composition specifically disclosed herein, such as a PL waxor a MOPL composition (hereinafter referred to as ‘Composition X’):

(i) Tablet 1 mg/tablet ‘Composition X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Composition X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Composition X’ 10.0 Colloidal silicon dioxide1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Composition X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Composition X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Composition X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt. % ‘Composition X’   5% Carbomer 934 1.25%Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben  0.2%Purified water q.s. to 100 g

(viii) Topical Gel 2 wt. % ‘Composition X’   5% Methylcellulose   2%Methyl paraben  0.2% Propyl paraben 0.02% Purified water q.s. to 100 g

(ix) Topical Ointment wt. % ‘Composition X’   5% Propylene glycol   1%Anhydrous ointment base  40% Polysorbate 80   2% Methyl paraben 0.2%Purified water q.s. to 100 g

(x) Topical Cream 1 wt. % ‘Composition X’  5% White bees wax 10% Liquidparaffin 30% Benzyl alcohol  5% Purified water q.s. to 100 g

(xi) Topical Cream 2 wt. % ‘Composition X’ 5% Stearic acid 10%  Glycerylmonostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropylpalmitate 2% Methyl Paraben 0.2%   Purified water q.s. to 100 g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Composition X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

1. A process for efficiently providing a composition with high amountsof omega-3 phospholipids comprising: contacting immature fish roe with apolar solvent; extracting a lipid fraction from the immature fish roe,to provide a primarily polar lipid fraction comprising omega-3phospholipids; and removing the solvent from the lipid fraction, toprovide a solid polar lipid composition comprising omega-3phospholipids, wherein at least about 40% of the total fatty acids ofthe lipid fraction are omega-3 fatty acids, the fatty acids of thecomposition comprise docosahexaenoic acid (DHA) and eicosapentaenoicacid (EPA), and the ratio of DHA to EPA is at least about 1.3:1.
 2. Theprocess of claim 1 wherein the immature fish roe is immature herringroe, immature salmon roe, immature mackerel roe, immature menhaden roe,or a combination thereof.
 3. The process of claim 2 wherein the polarlipid composition comprises about 50 wt. % to about 100 wt. %phospholipids, and about 30-60 wt. % of the total fatty acids in thephospholipids are omega-3 fatty acids.
 4. The process of claim 3 whereinthe polar lipid composition comprises at least about 50 wt. % ofphosphatidylcholine, with respect to the total weight of thecomposition.
 5. The process of claim 4 wherein the polar lipidcomposition comprises DHA in an amount of about 20%-40% expressed as apercentage of total fatty acids in the composition and EPA in amount ofabout 6%-20% expressed as a percentage of total fatty acids in thecomposition.
 6. The process of claim 5 wherein the polar lipidcomposition comprises at least about 40 wt. % of phosphatidylcholine,with respect to the total weight of the composition.
 7. The process ofclaim 6 wherein the polar lipid composition comprisesphosphatidylcholine (PC) and phosphatidylethanolamine (PE), and theratio of PC to PE is at least about 7.5:1.
 8. The process of claim 7wherein the lipid composition comprises less than about 1 wt. % ofarachidonic acid.
 9. The process of claim 8 wherein the polar lipidcomposition is characterized by an amber color.
 10. A compositionprepared by the process of claim
 9. 11. The process of claim 1 furthercomprising combining the polar lipid composition with an oil from asource other than the source of the polar lipid composition.
 12. Theprocess of claim 11 wherein the oil comprises vegetable oil, krill oil,microbial oil, fish oil, or a combination thereof.
 13. The process ofclaim 12 further comprising formulating a dietary supplement,nutritional supplement, pharmaceutical product, or food product with thepolar lipid composition as an ingredient.
 14. The process of claim 12further comprising formulating an oral delivery vehicle with the polarlipid composition as an ingredient.
 15. A lipid composition comprising aphospholipid wax and a carrier oil, wherein: the phospholipid waxcomprises at least about 35 wt. % of phosphatidylcholine; about 40-55wt. % the fatty acids of the phospholipid wax are docosahexaenoic acid(DHA) and eicosapentaenoic acid (EPA), and the ratio of DHA to EPA is atleast about 1.5:1; the carrier oil comprises mono-, di- ortriglycerides; fatty acid ethyl esters; free fatty acids; phospholipids,or a combination thereof; and the lipid composition comprises about 20wt. % to about 90 wt. % of the phospholipid wax and about 10 wt. % toabout 80 wt. % of the carrier oil; wherein the combination of thephospholipid wax and the carrier oil forms a viscous fluid oil.
 16. Thelipid composition of claim 15 wherein the ratio of DHA to EPA in thephospholipid wax is about is about 2.2:1 to about 3.5:1.
 17. The lipidcomposition of claim 15 wherein the phospholipid wax is a fish roeextract and the carrier oil is from a source other than fish roe,wherein the source other than fish roe is fish oil, vegetable oil, hilloil, microbial oil, or a combination thereof.
 18. An oral deliveryvehicle or functional food product comprising the lipid composition ofclaim
 15. 19. A method comprising administering a lipid composition ofclaim 15 to a subject to provide a phospholipid composition to thesubject wherein the ratio of DHA to EPA is at least about 2:1.
 20. Asolid phospholipid-rich lipid composition comprising greater than about60% phospholipids, wherein about 40-55 wt. % the fatty acids of thephospholipids are docosahexaenoic acid (DHA) and eicosapentaenoic acid(EPA), the ratio of DHA to EPA is about 1.5:1 to about 3.5:1, and thelipid composition is substantially stable to oxidation.
 21. The solidphospholipid-rich lipid composition of claim 20 wherein the ratio of DHAto EPA is about 2.2:1 to about 2.8:1 DHA:EPA.
 22. A process of producinga solid phospholipid-rich lipid composition with a high content of DHAcomprising: contacting a fish or fish by-product with a polar solvent toform a mixture with a polar solvent fraction; removing the solvent fromthe polar solvent fraction to provide a solid phospholipid-rich lipidcomposition comprising greater than about 60% phospholipids, wherein thecomposition comprises at least about 40 wt. % of phosphatidylcholine,about 40-55 wt. % the total fatty acids of the phospholipids aredocosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and theratio of DHA to EPA is about 1.5:1 to about 3.5:1, wherein the solidphospholipid lipid composition is substantially stable to oxidation. 23.An emulsion comprising water and a solid polar lipid compositionprepared by the process of claim 1, wherein the phospholipid waxcomposition increases the oxidative stability of the emulsion.
 24. Theemulsion of claim 23 wherein the emulsion is an oil-in-water emulsionand the emulsion is formed with about 0.1 wt. % to about 5 wt. % of thephospholipid fraction.
 25. The emulsion of claim 23 wherein the emulsionis a water-in-oil emulsion and the emulsion is formed with about 90 wt.% to about 98 wt. % of the phospholipid fraction.
 26. A compositioncomprising a solid polar lipid composition prepared by the process ofclaim 1 and a phospholipid extract from fish milt.
 27. The compositionof claim 26 further comprising a fish protein, a fish proteinhydrolysate, or fish protein-derived amino acids.